Collagen is the most abundant protein in vertebrates, occurring in virtually every tissue, including skin, tendon, bone, blood vessel, cartilage, ligament, and teeth. Collagen serves as the fundamental structural protein for vertebrate tissues. Collagen abnormalities are associated with a wide variety of human diseases, including arthritis, rheumatism, brittle bones, atherosclerosis, cirrhosis, and eye cataracts. Collagen is also critically important in wound healing. Increased understanding of the structure of collagen, and of how its structure affects its stability, facilitates the development of new treatments for collagen-related diseases and improved wound healing treatments.
Collagen is a fibrous protein that can exist in a variety of related forms. Mammals produce at least 17 distinct polypeptide chains that combine to form at least 10 variants of collagen. In each of these variants, the polypeptide chains of collagen are composed of approximately 300 repeats of the sequence X-Y-Gly, where X is often a proline (Pro) residue and Y is often a 4(R)-hydroxyproline (Hyp) residue. In connective tissue (such as bone, tendon, cartilage, ligament, skin, blood vessels, and teeth), individual collagen molecules are wound together in tight triple helices. These helices are organized into fibrils of great tensile strength, Jones & Miller, J. Mol. Biol., 218:209-219 (1991). Varying the arrangements and cross linking of the collagen fibrils enables vertebrates to support stress in one-dimension (tendons), two-dimensions (skin), or three-dimensions (cartilage).
In vertebrates, the collagen polypeptide is translated with the typical repeat motif being ProProGly. Subsequently, in vivo, the hydroxylation of Pro residues is performed enzymatically after collagen biosynthesis but before the chains begin to form a triple helix. Thus, hydroxylation could be important for both collagen folding and collagen stability. The hydroxyl group of Hyp residues has long been known to increase the thermal stability of triple-helical collagen, Berg and Prockop, Biochem. Biophys. Res. Comm., 52:115-120 (1973). For example, the melting temperature of a triple helix of (ProHypGly).sub.10 chains is 58.degree. C., while that of a triple helix of (ProProGly).sub.10 chains is only 24.degree. C., Sakakibara et al., Biochem. Biophys. Acta, 303:198-202 (1973). In addition, the rate at which (ProHypGly).sub.10 chains fold into a triple helix is substantially greater than the corresponding rate for (ProProGly).sub.10 chains, Chopra and Ananthanarayanan, Proc. Natl. Acad. Sci. USA, 79:7180-7184 (1982). The molecular basis for these observed effects is, however, not clear.
Molecular modeling based on the structure of triple-helical collagen and conformational energy calculations suggest that hydrogen bonds cannot form between the hydroxyl group of Hyp residues and any main chain groups of any of the collagen molecules in the same triple helix, Okuyama et al., J. Mol. Biol., 152:247-443 (1981). Several models include the hypothesis that hydroxyproline increases the stability of collagen as a result a bridge of water molecules formed between the hydroxyl group and a main chain carbonyl group. For reviews of observations advancing this hypothesis, see: Suzuki et al., Int. J. Biol. Macromol., 2:54-56 (1980), and Nemethy, in Collagen, published by CRC press (1988), and the references cited therein.
However, there exists experimental evidence that is inconsistent with the bridging water molecule model. For example, the triple helices of (ProProGly).sub.10 and (ProHypCly).sub.10 were found to be stable in 1,2-propanediol, and Hyp residues conferred added stability in these anhydrous conditions, Engel et al., Biopolymers, 16:601-622 (1977), suggesting that water molecules do not play a part in the added stability of (ProHypcly).sub.10. In addition, heat capacity measurements are inconsistent with collagen having more than one bound water per six Gly-X-Y units, Hoeve and Kakivaya, J. Phys. Chem., 80:754-749 (1976). Accordingly, there exists no prior definitive demonstration of the mechanism by which the hydroxyproline residues stabilizes collagen triplexes.
A better understanding of how the structure of collagen contributes to its stability would facilitate the design of a collagen or collagen mimics having improved stability. A high stability collagen substitute could advance the development of improved wound healing treatments.
In recent years, there have been exciting developments in wound healing, including the development of tissue engineering and tissue welding. For example, autologous epidermal transplantation for the treatment of burns was a significant advance in tissue engineering. Tissue engineering has also led to the development of several types of artificial skin, some of which employ human collagen as a substrate. However, a major problem associated with this treatment is the fragility of these grafts during and after surgery.
Tissue welding is a wound healing technique in which a laser is used to thermally denature the collagen in the skin at the periphery of a wound. The wound is reannealed by permitting the renaturation of the collagen. In the case of large wounds, a "filler" or solder is required to effect reannealing of the wound. Various materials, including human albumin, have been used as solders for this purpose. A good solder is resilient and is non-immunogenic and should preferably be capable of interaction with native collagen in adjacent sites.
Collagen is also used for a variety of other medical purposes. For example, collagen is used in sutures which can be naturally degraded by the human body and thus do not have to be removed following recovery. A sometimes limiting factor in the design of collagen sutures is the strength of the collagen fibers. A collagen variant or mimic having a greater strength would aid in the usage of such collagen sutures by relieving this limitation.
What is needed in the art is a novel collagen having increased stability for use in artificial skin, as a solder in tissue welding, and as a general tool for use in the design of medical constituents.
Fluoroproline (Flp) was synthesized by Gottleib et al., Biochemistry, 4:11:2507-2513 (1965) in both R and S stereoisomers. Gottleib et al. claimed to have incorporated both isomers into collagen by a biosynthetic route, but that claim was later refuted by Takeuchi et al., Biochem. Biophys. Acta, 175:156-164 (1969), Takeuchi and Prockop, Biochem. Biophys. Acta, 175:142-155 (1969), and Uitto and Prockop, Arch. Biochem. Biophys., 181:293-299 (1977). Because Gottleib et al. used biosynthesis, to the extent that Flp was incorporated at all into the resulting collagen molecules, it would have been incorporated randomly into the polypeptide in place of some random proline residues. There is, of course, no codon specific for Flp. The Flp was also a racemic mixture of both stereoisomers further randomizing the nature of the proteins produced, if the Flp was incorporated at all, which is significantly in doubt. Others have studied the chemical properties of Flp without incorporating it into a larger polypeptide, Gerig and McLeod, J. Am. Chem. Soc., 98:3970-3975 (1976).